Genetic midification of plants for enhanced resistance and decreased uptake of heavy metals

ABSTRACT

The present invention relates to a method of producing transformants with enhanced resistance and decreased uptake of heavy metals, and a plant transformed with a P type ATPase ZntA gene that pumps out heavy metals from the cells. The transformants show better growth than wild type in environment contaminated with heavy metals and have lower heavy metal contents than wild type plants. Therefore, this method of transforming plants with ZntA or biologically active ZntA-like heavy metal pumping ATPases can be useful for developing plants for phytoremediation and also for a safe crop that has resistance to heavy metals and low heavy metal contents.

BACKGROUND OF THE INVENTION

[0001] (a) Field of the Invention

[0002] The present invention relates to a method of producingtransformants with enhanced heavy metal resistance. More particularly,the present invention relates to transgenic plants that have an improvedgrowth but decreased heavy metal contents when grown in environmentcontaminated with heavy metals, thus this method can be used fordeveloping plants for phytoremediation and also for developing safecrops.

[0003] (b) Description of the Related Art

[0004] Heavy metals are major environmental toxicants, which causereactive oxidation species generation, DNA damage, and enzymeinactivation by binding to active sites of enzymes in cells.

[0005] Contamination of the environment with heavy metals has increaseddrastically due to industrialization. By the early 1990s, the worldwideannual release had reached 22,000 tons of cadmium, 954,000 tons ofcopper, 796,000 tons of lead, and 1,372,000 tons of zinc (Alloway B J &Ayres D C (1993) Principles of environmental pollution. Chapman andHall, London). The soils contaminated with heavy metal inhibit normalplant growth and cause contamination of foodstuffs. Many heavy metalsare very toxic to human health and carcinogenic at low concentrations.Therefore removal of heavy metals from the environment is an urgentissue.

[0006] Studies for removing heavy metals from soil are very activelyprogressing worldwide. Traditional methods of dealing with soilcontaminants include physical and chemical approaches, such as theremoval and burial of the contaminated soil, isolation of thecontaminated area, fixation (chemical processing of the soil toimmobilize the metals), and leaching using an acid or alkali solution(Salt D E, Blaylock M, Kumar N P B A, Viatcheslav D, Ensley B D, et al.(1995). Phytoremediation: a novel strategy for the removal of toxicmetals from the environment using plants. Bio-Technology 13,468-74;Raskin I, Smith R D, Salt D E. (1997) Phytoremediation of metals: usingplants to remove pollutants from the environment. Curr. Opin.Biotechnol. 8, 221-6). These methods, however, are costly andenergy-intensive processes.

[0007] Phytoremediation has recently been proposed as a low-cost,environment-friendly way to remove heavy metals from contaminated soils,and is a relatively new technology for cleanup of contaminated soil thatuses general plants, specially bred plants, or transgenic plants toaccumulate, remove, or detoxify environmental contaminants. Thephytoremediation technology is divided into phytoextraction,rhizofiltration, and phytostabilization.

[0008] Phytoextraction is a method using metal-accumulating plants toextract metals from soil into the harvestable parts of the plants;rhizofiltration is a method using plant roots to remove contaminantsfrom polluted aqueous streams; and phytostabilization is thestabilization of contaminants such as toxic metals in soils to preventtheir entry into ground water, also with plants (Salt et al.,Biotechnology 13(5): 468-474, 1995).

[0009] Examples of phytoremediation are methods using the plants ofLarrea tridentate species that are particularly directed at thedecontamination of soils containing copper, nickel, and cadmium (U.S.Pat. No. 5,927,005), and a method using Brassicaceae family (Baker etal., New Phytol. 127:61-68, 1994).

[0010] In addition, phytoremediation using transgenic plants that aregenerated by introducing genes having resistant activity for heavymetals have been attempted. Examples of heavy metal resistant genes areCAX2 (Calcium exchanger 2), cytochrome P450 2E1, NtCBP4 (Nicotianatabacum calmodulin-binding protein), GSHII (glutathione synthetase),merB (organomercurial lyase), and MRT polypeptide (metal-regulatedtransporter polypeptide).

[0011] CAX2 (Calcium exchanger 2), isolated from Arabidopsis thaliana,accumulates heavy metals including cadmium and manganese in plants(Hirschi et al., Plant Physiol. 124:125-134, 2000). Cytochrome P450 2E1uptakes and decomposes organic compounds such as trichloroethylene (DotySL et al., Proc. Natl. Acad. Sci. USA 97:6287-6291, 2000). Nicotianatabacum transformed with NtCBP4 has resistant activity for nickel (Araziet al., Plant J. 20:171-182, 1999), GSHII accumulates cadmium (Liang etal., Plant Physiol. 119:73-80,1999), merB detoxifies organic mercury(Bizily et al., Proc. Natl. Acad. Sci. USA 96:6808-6813, 1999), and MRTpolypeptide removes heavy metals including cadmium, zinc, and manganesefrom contaminated soil (U.S. Pat. No. 5,846,821).

[0012] However, the transgenic plants generated by introducing theabove-mentioned genes have limitations in growth due to accumulation ofheavy metals, and they can produce contaminated fruits and crops, whengrown in contaminated soil. Therefore, there is a need for plants thathave a lower uptake of heavy metals than the wild type, and thatmaintain healthy growth even in an environment contaminated with heavymetals.

SUMMARY OF THE INVENTION

[0013] It is an object of the invention to provide a gene, whenexpressed in plants, that confers heavy metal resistance and that caninhibit accumulation of heavy metals.

[0014] It is a further object of the invention to provide a recombinantvector harboring a heavy metal resistant gene.

[0015] It is a further object of the invention to provide a method forproducing transformants that have heavy metal resistance and thataccumulate less heavy metals than wild type plants.

[0016] It is a further object of the invention to provide transformantsthat have heavy metal resistance and that accumulate less heavy metalsthan wild type plants.

[0017] It is a further object of the invention to provide a method oftransforming a polluted area into an environmentally friendly space.

[0018] To accomplish the aforementioned objects, the invention providesa recombinant vector containing a coding sequence for a heavymetal-transporting P type ATPase, wherein the coding sequence isoperably linked to and under the regulatory control of aplant-expressible transcription and translation regulatory sequence.

[0019] Also, the invention provides a transgenic plant, or partsthereof, each transformed with a recombinant vector.

[0020] Also, the invention provides a transgenic plant cell.

[0021] Also, the invention provides a transgenic plant, stablytransformed with a recombinant vector.

[0022] Also, the invention provides a recombinant vector comprising acoding sequence for a heavy metal-transporting P type ATPase, ZntA ofSEQ ID NO: 1;

[0023] wherein the coding sequence is operably linked to and under theregulatory control of a plant-expressible transcription and translationregulatory sequence; and

[0024] wherein the ZntA contains an approximately 100 amino acid residueN-terminal extension domain, a first transmembrane spanning domain, asecond transmembrane spanning domain containing a putative cationchannel motif CPX domain, a third transmembrane spanning domain, a firstcytoplasmic domain, a second cytoplasmic domain, and a C-terminal domain

[0025] Also, the invention provides a recombinant vector comprising acoding sequence for a heavy metal-transporting P type ATPase, ZntAwherein the coding sequence is operably linked to and under theregulatory control of a plant-expressible transcription and translationregulatory;

[0026] wherein the ZntA contains an approximately 100 amino acid residueN-terminal extension domain, a first transmembrane spanning domain, asecond transmembrane spanning domain containing a putative cationchannel motif CPX domain, a third transmembrane spanning domain, a firstcytoplasmic domain, a second cytoplasmic domain, and a C-terminaldomain; and

[0027] wherein each of the domains of the coding sequence shares atleast about 50% homology with a same domain of SEQ ID NO:1.

[0028] Also, the invention provides a method of producing a transgenicplant with enhanced resistance to heavy metals comprising:

[0029] (a) preparing an expression construct comprising a sequenceencoding a heavy metal-transporting P type ATPase, operably linked toand under the regulatory control of a plant-expressible transcriptionand translation regulatory sequence;

[0030] (b) preparing a recombinant vector harboring the expressionconstruct; and

[0031] (c) introducing the expression construct of the recombinantvector into a plant cell or plant tissue to produce a transgenic plantcell or transgenic plant tissue

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 represents the map of the recombinant vector pEZG.

[0033]FIG. 2 shows plasma membrane localization of ZntA proteinexpressed in Arabidopsis protoplasts.

[0034]FIG. 3 is a Western blot photograph showing membrane localizationof ZntA protein expressed in Arabidopsis protoplast.

[0035]FIG. 4 represents the map of recombinant vector PBI121/zntA.

[0036]FIG. 5 is a Northern blot photograph showing expression of zntAmRNA in Arabidopsis.

[0037]FIG. 6 shows the enhanced growth of zntA-transgenic plants overthat of wild type in a medium containing lead.

[0038]FIG. 7 shows the enhanced growth of zntA-transgenic plants overthat of wild type in a medium containing cadmium.

[0039]FIG. 8 is a graph showing the weight of zntA-transgenic pantscultivated in media containing heavy metals.

[0040]FIG. 9 is a graph showing the chlorophyll contents ofzntA-transgenic and wild type plants, grown in media containing heavymetals.

[0041]FIG. 10 is a graph showing the heavy metal contents ofzntA-transgenic and wild type plants, grown in media containing heavymetals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] As used herein, the term “P type ATPase” refers to a transporterthat transports a specific material by using energy from ATP hydrolysisand that forms a phosphorylated intermediate. More particularly, the Ptype ATPase is a heavy metal-transporting ATPase. The heavy metal is ametal element having a specific gravity over 4 including arsenic (As),antimony (Sb), lead (Pb), mercury (Hg), cadmium (Cd), chrome, tin (Sn),zinc, barium (Ba), nickel (Ni), bismuth (Bi), cobalt (Co), manganese(Mn), iron (Fe), copper (Cu), and vanadium (V).

[0043] ZntA is a P type ATPase of E. coli (Rensing C, Mitra B, Rosen BP. (1997) Proc. Natl. Acad. Sci. USA. 94,14326-31; Sharma, R., Rensing,C., Rosen, B. P., Mitra, B. (2000) J Biol. Chem. 275,3873-8) which pumpsPb(II)/Cd(II)/Zn(II) across the plasma membrane.

[0044] P-type ATPases typically have 2 large cytoplasmic domains and 6transmembrane domains. ZntA has similar domains, and in addition, 2 moretransmembrane helixes at N-terminus and N-terminal extension of about100 amino acids containing CXXC motif. The first large cytoplasmicdomain of ZntA is about 145 amino acid long and involved in hydrolysisof phosphointermediate, and the second large cytoplasmic domain is 280amino acid long and has a phosphorylation motif. We denote the 4transmembrane helixes of the N-terminal side as the first transmembranespanning domain. The 2 transmembrane helixes between the 2 largecytoplasmic domains is denoted as the second transmembrane spanningdomain. This domain contains a putative cation channel motif CPX domain.The transmembrane helixes between the second large cytoplasmic domainand the c-terminus is denoted as the third transmembrane spanningdomain. The cytoplasmic domain following the third transmembranespanning domain is denoted as the C-terminal domain of ZntA.

[0045] The term “homology” refers to the sequence similarity between 2DNA or protein molecules. “Biologically active ZntA-like heavy metalpumping ATPases” are coded by DNA sequences which have at least 50%homology to ZntA, and have heavy metal pumping activity. Biologicallyactive ZntA-like heavy metal pumping ATPases include zinc-transportingATPase (NC_(—)000913), zinc-transporting ATPase (NC_(—)002655), heavymetal-transporting ATPase (NC_(—)003198), P-type ATPase family(NC_(—)003197), cation transporting P-type ATPase from Mycobacteriumlepraed (GenBank #Z46257), and many others.

[0046] A “heavy metal resistance protein” is a protein capable ofmediating resistance to at least one heavy metal, including, but notlimited to, lead, cadmium, and zinc. An example of a heavy metalresistance protein is ZntA protein of SEQ ID NO:1.

[0047] The term “plant-expressible” means that the coding sequence isoperably linked to and under the regulatory control of a transcriptionand translation regulatory sequence that can be efficiently expressed byplant cells, tissues, parts and whole plants.

[0048] “Plant-expressible transcriptional and translational regulatorysequences” are those which can function in plants, plant tissues, plantparts and plant cells to effect the transcriptional and translationalexpression of the target sequence with which they are associated.Included are 5′ sequences of a target sequence to be expressed, whichqualitatively control gene expression (turn gene expression on or off inresponse to environmental signals such as light, or in a tissue-specificmanner); and quantitative regulatory sequences which advantageouslyincrease the level of downstream gene expression. An example of asequence motif that serves as a translational control sequence is thatof the ribosome binding site sequence. Polyadenylation signals areexamples of transcription regulatory sequences positioned downstream ofa target sequence, and there are several that are well known in the artof plant molecular biology.

[0049] A “transgenic plant” is one that has been genetically modified,unlike the wild type plants. Transgenic plants typically expressheterologous DNA sequences, which confer the plants with charactersdifferent from that of wild type plants. As specifically exemplifiedherein, a transgenic plant is genetically modified to contain andexpress at least one heterologous DNA sequence that is operably linkedto and under the regulatory control of transcriptional control sequenceswhich function in plant cells or tissue, or in whole plants.

[0050] The present invention provides a plant-expressible expressionconstruct containing a coding sequence for a heavy metal-transportingATPase protein. The coding sequence is operably linked to and under theregulatory control of a plant-expressible transcription and translationregulatory sequence. The heavy metals include arsenic (As), antimony(Sb), lead (Pb), mercury (Hg), cadmium (Cd), chrome, tin (Sn), zinc,barium (Ba), nickel (Ni), bismuth (Bi), cobalt (Co), manganese (Mn),iron (Fe), copper (Cu) and vanadium (V).

[0051] The expression construct includes a promoter, a heavymetal-transporting P type ATPase gene, and a transcriptional terminator.The suitable plant-expressible promoters include the ³⁵S or 19Spromoters of Cauliflower Mosaic Virus; the nos (nopaline synthase), ocs(octopine synthase), or mas (mannopine synthase) promoters ofAgrobacterium tumefaciens Ti plasmids; and others known to the art.

[0052] The heavy metal-transporting ATPase gene of the present inventionprefers genes encoding ZntA (SEQ ID NO:1) or biologically activeZntA-like heavy metal pumping ATPase genes, which have at least 50%homology to ZntA, and which code for proteins with heavy metal pumpingactivities.

[0053] The heavy metal-transporting ATPase gene of the present inventionalso prefers DNA sequences containing an approximately 100 amino acidresidue N-terminal extension domain, a first transmembrane spanningdomain, a second transmembrane spanning domain containing a putativecation channel motif CPX domain, a third transmembrane spanning domain,a first cytoplasmic domain, a second cytoplasmic domain, and aC-terminal domain of ZntA, or DNA sequences which share at least 50%homology with abovementioned domains of the biologically activeZntA-like heavy metal pumping ATPase genes.

[0054] The expression construct of the present invention may furthercontain a marker allowing selection of transformants in the plant cellor showing a localization of a target protein. The examples of a markerare genes carrying resistance to an antibiotic such as kanamycin,hygromycin, gentamicin, and bleomycin; and genes coding GUS(α-glucuronidase), CAT (chloramphenicol acetyltransferase), luciferase,and GFP (green fluorescent protein). The marker allows for selection ofsuccessfully transformed plant cells growing in a medium containingcertain antibiotics because they will carry the expression constructwith the resistance gene to the antibiotic.

[0055] Also, the invention provides a recombinant vector comprising theexpression construct. The recombinant vector comprises a backbone of thecommon vector and the expression construct. The common vector ispreferably selected from the group consisting of pROKII, pBI76, pET21,pSK(+), pLSAGPT, pBI121, and PGEM. Examples of the prepared recombinantvector are PBI121/zntA and pEZG. PBI121/zntA comprises a backbone ofPBI121, CMV ³⁵S promoter, zntA gene, and nopaline synthase terminator;and pEZG comprises a backbone of pUC, CMV ³⁵S promoter, zntA gene, greenfluorescence protein, and nopaline synthase terminator.

[0056] Also, the present invention provides a transformant containingthe expression construct. The transformant contains a DNA sequenceencoding a heavy metal-transporting P type ATPase, wherein the codingsequence is operably linked to and under the regulatory control of atranscription and translation regulatory sequence.

[0057] The transformant is preferably a plant, and more preferably aplant, parts thereof, and plant cell. The plant parts include a seed.The plants are herbaceous plants and trees, and they include floweringplants, garden plants, an onion, a carrot, a cucumber, an olive tree, asweet potato, a potato, a cabbage, a radish, lettuce, broccoli,Nicotiana tabacum, Petunia hybrida, a sunflower, Brassica juncea, turf,Arabidopsis thaliana, Brassica campestris, Betula platyphylla, a poplar,a hybrid poplar, and Betula schmidtii.

[0058] Techniques for generating transformants are well known. Anexample is Agrobacterium tumefaciens-mediated DNA transfer. Preferably,recombinant A. tumefaciens generated by electroporation, micro-particleinjection, or with a gene gun can be used.

[0059] In addition, the invention provides a method of producing atransgenic plant with enhanced resistance to heavy metals, comprising:

[0060] (a) preparing an expression construct comprising aplant-expressible sequence encoding a heavy metal-transporting P typeATPase, operably linked to and under the regulatory control of atranscription and translation regulatory sequence;

[0061] (b) preparing a recombinant vector harboring the expressionconstruct; and

[0062] (c) introducing the expression construct of the recombinantvector into a plant cell or plant tissue to produce a transgenic plantcell or transgenic plant tissue.

[0063] The method of producing a transgenic plant further comprises astep: (d) regenerating a transgenic plant from the transgenic plant cellor transgenic plant tissue of step (c).

[0064] In the present invention, ZntA protein was expressed in theplasma membrane (FIGS. 2 and 3). Moreover, zntA-transgenic Arabidopsisplants showed enhanced resistance to lead and cadmium, and the contentof lead and cadmium was lower than in a wild-type plant.

[0065] Therefore, zntA-transgenic plants or plants transformed with agene encoding biologically active ZntA-like heavy metal pumping ATPasescan grow in an environment contaminated with heavy metals, and thistechnique can be useful for generating crop plants with decreased uptakeof harmful heavy metals. Since harmful heavy metals can be introducedinto farmland inadvertently, for example, due to the yellow sandphenomenon or by natural disaster, heavy metal pumping transgenic cropplants can be a safe choice for health-concerned consumers.

[0066] The following examples are provided for illustrative purposes andare not intended to limit the scope of the invention as claimed herein.Any variations in the exemplified compositions and methods which occurto the skilled artisan are intended to fall within the scope of thepresent invention.

EXAMPLE 1. Isolation of ZntA Gene

[0067]Escherichia coli K-12 was obtained from the Korean Collection forType Cultures of the Korea Research Institute of Bioscience andBiotechnology, and a zntA gene was cloned.

[0068] zntA was isolated by PCR using genomic DNA of Escherichia coliK-12 strain as a template. PCR was performed with a primer set of SEQ IDNO:2, SEQ ID NO:3, and 2.2 kb of PCR product, and zntA of SEQ ID NO:1was obtained. The sequence of the PCR product was analyzed and the PCRproduct was cloned into a pGEM-T easy vector to produce pGEM-T/zntA.

EXAMPLE 2. Expression of ZntA Protein

[0069] A zntA gene was, introduced into Arabidopsis protoplasts, andlocalization of ZntA protein was investigated.

[0070] (2-1) Preparation of Arabidopsis Protoplasts

[0071] Arabidopsis protoplasts were prepared as described (Abel S,Theologis A (i 994) Transient transformation of Arabidopsis leafprotoplasts: a versatile experimental system to study gene expression.Plant J. 5, 421-7).

[0072] Seeds of Arabidopsis were placed into an antiseptic solution(distilled water: chlorox: 0.05% triton X-100=3:2:2), shaken for 20-30seconds, and incubated at room temperature for 5-10 mins. The seeds werethen rinsed five times with distilled water.

[0073] The Arabidopsis seeds were incubated in 100 ml of a liquidsolution (Murashige & Skoog medium; MSMO, pH 5.7-5.8) containingvitamins, Duchefa 4.4 g/L, sucrose 20 g/L, MES (2-(N-Morpholino)Ethanesulfonic acid, Sigma) 0.5 g/L, while agitating at 120 rpm under a16/8 hr (light/dark) cycle, at 22° C. for 2-3 weeks.

[0074] The 2-3 week-old whole plants were chopped with a razor blade to5-10 mm² pieces. These leaf fragments were transferred to an enzymesolution (1% cellulase R-10, 0.25% marcerozyme R-10, 0.5 M mannitol, 10mM MES, 1 mM CaCl₂, 5 mM β-mercaptoethanol, and 0.1% BSA, pH 5.7-5.8),vacuum-infiltrated for 10 min, and then incubated in the dark at 22° C.for 5 hours with gentle agitation at 50-75 rpm. The released protoplastswere filtered through a 100 μm mesh (Sigma S0770, USA), purified using a21% sucrose gradient by centrifugation at 730 rpm for 10 min, and thensuspended in 20 ml of W5 solution (154 mm NaCl, 125 mM CaCl₂, 5 mM KCl,5 mM glucose, and 1.5 mM MES, pH 5.6) and centrifuged again at 530 rpmfor 6 min. The pellected protoplasts were re-suspended in W5 solutionand kept on ice.

[0075] (2-2) Preparation of Vector

[0076] pGEM-T/zntA DNA was cut with BamHI restriction enzyme and zntAgenes were extracted (QIAGEN Gel extraction kit). The zntA genes wereplaced under the control of a Cauliflower Mosaic Virus 35S promoter,fused with and then inserted into a pUC-GFP vector containing GreenFluorescent Protein (GFP) and nopaline synthase terminator (NOS), tothereby produce pEZG.

[0077] (2-3) Preparation of Vector for H⁺ Pumping Gene

[0078] A hydrogen ion pump gene of Arabidopsis, AHA2 cDNA (Gene Bank:P19456), was amplified by PCR. Primers for PCR were polynucleotides ofSEQ ID NO:4 and SEQ ID NO:5. PCR conditions were as follows: 94° C., 30sec->45° C., 30 sec->72° C., 1 min, 50 cycles. The PCR product wasobtained as AHA2 cDNA.

[0079] A DsRed vector (Clontech, Inc.) was treated with BgIII/NotIrestriction enzyme and DsRed was obtained. The DsRed was inserted intothe opened smGFP vector with a BamHI/EcI136II restriction enzyme to326RFP. In addition, AHA2 cDNA was inserted at XmaI of the 326RFP vectorand 326RFP/AHA2 was prepared.

[0080] (2-4) Introduction of pEZG or 326RFP/AHA2 Into Protoplast

[0081] pEZG and 326RFP/AHA2 were introduced to the protoplasts preparedby EXAMPLE (2-1), and expression of foreign genes was confirmed.

[0082] The protoplast was centrifuged at 500 rpm for 5 min, and 5×10⁶/mlof the protoplast were suspended in a MaMg solution (400 mM mannitol, 15mM MgCl₂, 5 mM MES-KOH, pH 5.6). 300 μl of the suspension solution wasmixed with 10 μg of pEZG and 326RFP/AHA2 respectively, which was thenwas added to 300 μl of PEG (400 mM mannitol, 100 mM Ca(NO₃)₂, 40% PEG6000), and stored at RT for 30 min. The mixture was washed with 5 ml ofW5 solution, centrifuged at 500 rpm for 3 min, and a pellet wasobtained. The pellet was washed with 2 ml of W5 solution and incubatedin the dark at 22-25° C. After 24 hr, expression of GFP protein wasmonitored and images were captured with a cooled charge-coupled devicecamera using a Zeiss Axioplan fluorescence microscope. The filter setsused for the GFP were XF116 (exciter, 474AF20; dichroic, 500DRLP;emitter, 510AF23) (Omega, Inc., Brattleboro, Vt.). Data were thenprocessed using Adobe (Mountain View, Calif.) Photoshop software.

[0083]FIG. 2 shows a localization of ZntA protein fused with GFP inprotoplasts transformed with pEZG and 326RFP/AHA2, respectively. “a” iscontrol, “b” is AHA2 protein expressed in 326RFP/AHA2, “c” is ZntAprotein expressed in pEZG, and “d” is an overlapped picture of “b” and“c”. ZntA fused with GFP shows a green color due to GFP, and AHA2 fusedwith DsRed shows a red color due to DsRed.

[0084] In FIG. 2, ZntA fused with GFP was localized at the plasmamembrane in Arabidopsis protoplasts.

[0085] In addition, membrane and cytosol fractions were isolated fromArabidopsis protoplasts, and Western Blot was preformed using a GFPantibody as a probe. FIG. 3 is a Western Blot photograph, wherein “WT-C”is cytosol of wild-type Arabidopsis protoplasts, “WT-M” is membrane ofwild-type Arabidopsis protoplasts, “ZntA-C” is cytosol of Arabidopsisprotoplasts transformed with pEZG, and “ZntA-M” is membrane ofArabidopsis protoplasts transformed with pEZG. In FIG. 3, the GFPantibody cross-reacted only with membrane proteins extracted fromArabidopsis protoplasts transformed with pEZG, confirming that ZntAprotein was expressed in membrane.

EXAMPLE 3. Preparation of Transgenic Plants Expressing ZntA Protein.

[0086] (3-1) Arabidopsis

[0087] Arabidopsis plants were grown at 4° C. for 2 days, then they weregrown with a 16/8 hr (light/dark) photoperiod, at 22° C./18° C. for 3-4weeks until flower stalks were differentiated. The 1^(st) flower stalkwas removed, and the 2^(nd) flower stalk was used for transformation.

[0088] (3-2) pBI121/ZntA Vector

[0089] A zntA gene was inserted into the expression vector for theplant, preparing pBI121 and pBI121/zntA.

[0090] A GUS gene of pBI121 was removed by digesting with SmaI andEcI136II restriction enzymes, and a zntA gene prepared from thepGEM-T/zntA was inserted to pBI121, thereby preparing a pBI121/zntAvector (FIG. 4).

[0091] (3-3) Preparation of Transgenic Plants

[0092] pBI121/zntA vector DNA was isolated with a prep-kit (Qiagen) andintroduced to agrobacterium using electroporation. The agrobacterium(KCTC 10270BP) was cultured in YEP media (yeast extract 10 g, NaCl 5 g,pepton 10 g, pH 7.5) until index of O.D. reached 0.8-1.0. The culturesolution was centrifuged, cells were collected and suspended in MS media(Murashige & Skoog medium, 4.3 g/L, Duchefa) containing 5% sucrose, andSilwet L-77 (LEHLE SEEDS, USA) was added as a final concentration of0.01% just before transformation. For plant transformation, pBI121/zntAwas introduced into the Agrobacterium LBA4404 strain, which was thenused to transform Arabidopsis by a dipping method (Clough S J, and BentA F (1988), Floral dip: a simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana. Plant J. 16, 735-743).

EXAMPLE 4. Selection of Transformants

[0093] For selection of plant transformed with zntA genes, plants weregrown in solid Murashige-Skoog (MS) medium containing kanamycin (50mg/l). T2 or T3 generation seeds were used for the tests. Also, a pBI121vector was introduced to Arabidopsis and transformants (pBI121 plants)were selected. Seeds were obtained from wild-type Arabidopsis, pBI121plants, and pBI121/zntA plants, respectively.

[0094] To test the ZntA expression level, total RNA was isolated fromkanamycin-selected T2 plants and used for Northern Blot analysis. TotalRNA was extracted from Arabidopsis plants grown on the 1/2 MS (Murashige& Skoog medium, 2.15 g/L, Duchefa)-agar media for 3 weeks. SubsequentRNA preparation and northern hybridization followed the establishedmethod (Sambrook et al. (2001) Molecular Cloning: A laboratory manual(Third

[0095] Edition), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) with slight modifications.

[0096] The plant materials were frozen in liquid nitrogen andhomogenized with mortars and pestles. 1 ml of TRIzol reagent (Lifetechnology, USA) per 100 mg of tissue was added to the sample and after5 min incubation at RT, 0.2 ml of chloroform per 1 ml of TRIzol reagentwas added. After centrifugation at 10,000 g for 10 min at 4° C., theaqueous phase was taken and precipitated with 0.5 ml of isopropylalcohol per 1 ml of TRIzol reagent and quantified by UV spectroscopy.Total RNA was separated in a 10 formaldehyde-containing agarose gel andthen transferred onto a nylon membrane. After UV crosslinking,hybridization was carried out in a modified Church buffer (7% (w/v) SDS,0.5 M sodium phosphate (pH 7.2), 1 mM EDTA (pH 7.0)) at 68° C.overnight, with ³²P-labeled zntA probes. Membranes were washed once for10 min in 1×SSC, 0.1% SDS at room temperature, and twice for 10 min in0.5×SSC, 0.1% SDS at 68° C. The membrane was exposed to a phosphorimagerscreen (Fuji film) or x-ray film (Kodak). The mRNA expression levelswere analyzed by the Mac-BAS image-reader program. FIG. 5 is a NorthernBlot photograph showing expression of zntA mRNA in Arabidopsis.Transcription of zntA RNA was not observed in wild-type Arabidopsis andpBI121 plants, but it was observed in pBI121/zntA plants. EF1-a isconstitutively expressed in plants and its even levels indicated thatthe same amount of RNA was used for different samples.

EXAMPLE 5. Heavy Metals Resistance of Plant Transformed With ZntA Gene

[0097] Wild-type Arabidopsis plants and pBI121/zntA plants were grown in1/2 MS-agar media for 2 weeks and transferred 1/2 MS-liquid mediacontaining 70 μM cadmium or 0.7 mM lead. After 2 weeks, growth, weight,and heavy metal contents were measured.

[0098] (5-1) Growth of Plants

[0099]FIG. 6 shows the growth of wild-type and pBI121/zntA Arabidopsisplants grown in a medium containing lead. FIG. 7 shows wild-type andpBI121/zntA Arabidopsis plants grown in a medium containing cadmium.“WT” is wild-type Arabidopsis, “1” to “4” are pBI121/zntA plants. InFIGS. 6 and 7, pBI121/zntA plants grew better than the wild-type plants;their leaves were broader, greener, and their fresh weights were higherthan those of the wild types. These results indicate that the expressionof ZntA confers Pb(II)- and Cd(II)-resistance to the transgenic plants.

[0100] (5-2) Measurement of Biomass

[0101] Wild type and pBI121/zntA Arabidopsis plants were grown in 1/2MS-agar media for 2 weeks and then transferred to 1/2 MS-liquid mediasupported by small gravel with or without Cd (II) or Pb (II). Aftergrowing for an additional 2 weeks, the plants were harvested. They werewashed in an ice-cold 1 mM tartarate solution and blot-dried. The weightof the wild type and pBI121/zntA Arabidopsis plants were measured.

[0102]FIG. 8a is a graph showing the weight of wild type and pBI121/zntAplants grown in a medium containing lead, and FIG. 8b is a graph showingthe weight of wild type and pBI121/zntA plants grown in a mediumcontaining cadmium. The weight of pBI121/zntA plants was higher thanthat of the wild-type plants. These results indicate that plantsexpressing ZntA protein can grow better than wild type in soilcontaminated with heavy metals.

[0103] (5-3) Measurement of Chlorophyll Contents

[0104] For determination of chlorophyll contents, the leaves wereharvested and extracted with 95% ethanol for 20 min at 80° C. Absorbanceat 664 nm and 648 nm were measured and then the chlorophyll A and Bcontents were calculated as described (Oh S A, Park J H, Lee G I, Paek KH, Park S K, Nam H G (1997) Identification of three genetic locicontrolling leaf senescence in Arabidopsis thaliana. Plant J. 12,527-35).

[0105]FIG. 9a is a graph showing the chlorophyll contents of wild typeand zntA-transgenic plants grown in a medium containing lead, and FIG.9b is a graph showing the chlorophyll contents of wild type andzntA-transgenic plants grown in a medium containing cadmium. Thechlorophyll contents of zntA-transgenic plants were higher than those ofthe wild types.

[0106] (5-4) Measurement of the Heavy Metal Contents

[0107] We measured the content of Pb and Cd in control and ZntAoverexpressing plants grown in media containing heavy metals.pBI121/zntA plants were collected, weighed, and digested with 65% HNO₃at 200° C., overnight. Digested samples were diluted with 0.5 N HNO₃ andanalyzed using an atomic absorption spectrometer (AAS; SpectrAA-800,Varian).

[0108]FIG. 10 is a graph showing the heavy metal contents of wild typeand zntA-transgenic plants grown in media containing heavy metals. FIG.10a is the lead contents, and 10 b is the cadmium contents. Pb contentof pBI121/zntA plants varied between the lines, but it was consistentlylower than that of the wild type. Cd content in transgenic lines 1 and 3was lower than that in the control.

[0109] Thus, plants transformed with zntA or other biologically activeZntA-like heavy metal pumping ATPases can be grown in soil contaminatedwith heavy metals and have less uptake of heavy metals than wild typeplants. Since growing plants can hold contaminated soil and therebyreduce erosion of the soil, and since the zntA-transgenic plants cangrow better than wild type plants in soil contaminated by heavy metals,they can reduce migration of pollutants from the polluted area, therebyreducing contamination of groundwater by the pollutants. The presentinvention can also be applied to crop plants to produce low heavymetal-containing safe crop plants.

1 6 1 2199 DNA Escherichia coli zntA gene (1)..(2199) 1 atgtcgactcctgacaatca cggcaagaaa gcccctcaat ttgctgcgtt caaaccgcta 60 accacggtacagaacgccaa cgactgttgc tgcgacggcg catgttccag cacgccaact 120 ctctctgaaaacgtctccgg cacccgctat agctggaaag tcagcggcat ggactgcgcc 180 gcctgtgcgcgcaaggtaga aaatgccgtg cgccagcttg caggcgtgaa tcaggtgcag 240 gtgttgttcgccaccgaaaa actggtggtc gatgccgaca atgacattcg tgcacaagtt 300 gaatctgcgctgcaaaaagc aggctattcc ctgcgcgatg aacaggccgc cgaagaaccg 360 caagcatcacgcctgaaaga gaatctgccg ctgattacgc taatcgtgat gatggcaatc 420 agctggggtctggagcagtt caatcatccg ttcgggcaac tggcgtttat cgcgaccacg 480 ctggttgggctgtacccgat tgctcgtcag gcattacggt tgatcaaatc cggcagctac 540 ttcgccattgaaaccttaat gagcgtagcc gctattggtg cactgtttat tggcgcaacg 600 gctgaagctgcgatggtgtt gctgctgttt ttgattggtg aacgactgga aggctgggcc 660 gccagccgcgcgcgtcaggg cgttagcgcg ttaatggcgc tgaaaccaga aaccgccacg 720 cgcctgcgtaagggtgagcg ggaagaggtg gcgattaaca gcctgcgccc tggcgatgtg 780 attgaagtcgccgcaggtgg gcgtttgcct gccgacggta aactgctctc accgtttgcc 840 agttttgatgaaagcgccct gaccggcgaa tccattccgg tggagcgcgc aacgggcgat 900 aaagtccctgctggtgccac cagcgtagac cgtctggtga cgttggaagt gctgtcagaa 960 ccgggagccagcgccattga ccggattctg aaactgatcg aagaagccga agagcgtcgc 1020 gctcccattgagcggtttat cgaccgtttc agccgtatct atacgcccgc gattatggcc 1080 gtcgctctgctggtgacgct ggtgccaccg ctgctgtttg ccgccagctg gcaggagtgg 1140 atttataaagggctgacgct gctgctgatt ggctgcccgt gtgcgttagt tatctcaacg 1200 cctgcggcgattacctccgg gctggcggcg gcagcgcgtc gtggggcgtt gattaaaggc 1260 ggagcggcgctggaacagct gggtcgtgtt actcaggtgg cgtttgataa aaccggtacg 1320 ctgaccgtcggtaaaccgcg cgttaccgcg attcatccgg caacgggtat tagtgaatct 1380 gaactgctgacactggcggc ggcggtcgag caaggcgcga cgcatccact ggcgcaagcc 1440 atcgtacgcgaagcacaggt tgctgaactc gccattccca ccgccgaatc acagcgggcg 1500 ctggtcgggtctggcattga agcgcaggtt aacggtgagc gcgtattgat ttgcgctgcc 1560 gggaaacatcccgctgatgc atttactggt ttaattaacg aactggaaag cgccgggcaa 1620 acggtagtgctggtagtacg taacgatgac gtgcttggtg tcattgcgtt acaggatacc 1680 ctgcgcgccgatgctgcaac tgccatcagt gaactgaacg cgctgggcgt caaaggggtg 1740 atcctcaccggcgataatcc acgcgcagcg gcggcaattg ccggggagct ggggctggag 1800 tttaaagcgggcctgttgcc ggaagataaa gtcaaagcgg tgaccgagct gaatcaacat 1860 gcgccgctggcgatggtcgg tgacggtatt aacgacgcgc cagcgatgaa agctgccgcc 1920 atcgggattgcaatgggtag cggcacagac gtggcgctgg aaaccgccga cgcagcatta 1980 acccataaccacctgcgcgg cctggtgcaa atgattgaac tggcacgcgc cactcacgcc 2040 aatatccgccagaacatcac tattgcgctg gggctgaaag ggatcttcct cgtcaccacg 2100 ctgttagggatgaccgggtt gtggctggca gtgctggcag atacgggggc gacggtgctg 2160 gtgacagcgaatgcgttaag attgttgcgc aggagataa 2199 2 732 PRT Escherichia coli ZntAprotein (1)..(732) 2 Met Ser Thr Pro Asp Asn His Gly Lys Lys Ala Pro GlnPhe Ala Ala 1 5 10 15 Phe Lys Pro Leu Thr Thr Val Gln Asn Ala Asn AspCys Cys Cys Asp 20 25 30 Gly Ala Cys Ser Ser Thr Pro Thr Leu Ser Glu AsnVal Ser Gly Thr 35 40 45 Arg Tyr Ser Trp Lys Val Ser Gly Met Asp Cys AlaAla Cys Ala Arg 50 55 60 Lys Val Glu Asn Ala Val Arg Gln Leu Ala Gly ValAsn Gln Val Gln 65 70 75 80 Val Leu Phe Ala Thr Glu Lys Leu Val Val AspAla Asp Asn Asp Ile 85 90 95 Arg Ala Gln Val Glu Ser Ala Leu Gln Lys AlaGly Tyr Ser Leu Arg 100 105 110 Asp Glu Gln Ala Ala Glu Glu Pro Gln AlaSer Arg Leu Lys Glu Asn 115 120 125 Leu Pro Leu Ile Thr Leu Ile Val MetMet Ala Ile Ser Trp Gly Leu 130 135 140 Glu Gln Phe Asn His Pro Phe GlyGln Leu Ala Phe Ile Ala Thr Thr 145 150 155 160 Leu Val Gly Leu Tyr ProIle Ala Arg Gln Ala Leu Arg Leu Ile Lys 165 170 175 Ser Gly Ser Tyr PheAla Ile Glu Thr Leu Met Ser Val Ala Ala Ile 180 185 190 Gly Ala Leu PheIle Gly Ala Thr Ala Glu Ala Ala Met Val Leu Leu 195 200 205 Leu Phe LeuIle Gly Glu Arg Leu Glu Gly Trp Ala Ala Ser Arg Ala 210 215 220 Arg GlnGly Val Ser Ala Leu Met Ala Leu Lys Pro Glu Thr Ala Thr 225 230 235 240Arg Leu Arg Lys Gly Glu Arg Glu Glu Val Ala Ile Asn Ser Leu Arg 245 250255 Pro Gly Asp Val Ile Glu Val Ala Ala Gly Gly Arg Leu Pro Ala Asp 260265 270 Gly Lys Leu Leu Ser Pro Phe Ala Ser Phe Asp Glu Ser Ala Leu Thr275 280 285 Gly Glu Ser Ile Pro Val Glu Arg Ala Thr Gly Asp Lys Val ProAla 290 295 300 Gly Ala Thr Ser Val Asp Arg Leu Val Thr Leu Glu Val LeuSer Glu 305 310 315 320 Pro Gly Ala Ser Ala Ile Asp Arg Ile Leu Lys LeuIle Glu Glu Ala 325 330 335 Glu Glu Arg Arg Ala Pro Ile Glu Arg Phe IleAsp Arg Phe Ser Arg 340 345 350 Ile Tyr Thr Pro Ala Ile Met Ala Val AlaLeu Leu Val Thr Leu Val 355 360 365 Pro Pro Leu Leu Phe Ala Ala Ser TrpGln Glu Trp Ile Tyr Lys Gly 370 375 380 Leu Thr Leu Leu Leu Ile Gly CysPro Cys Ala Leu Val Ile Ser Thr 385 390 395 400 Pro Ala Ala Ile Thr SerGly Leu Ala Ala Ala Ala Arg Arg Gly Ala 405 410 415 Leu Ile Lys Gly GlyAla Ala Leu Glu Gln Leu Gly Arg Val Thr Gln 420 425 430 Val Ala Phe AspLys Thr Gly Thr Leu Thr Val Gly Lys Pro Arg Val 435 440 445 Thr Ala IleHis Pro Ala Thr Gly Ile Ser Glu Ser Glu Leu Leu Thr 450 455 460 Leu AlaAla Ala Val Glu Gln Gly Ala Thr His Pro Leu Ala Gln Ala 465 470 475 480Ile Val Arg Glu Ala Gln Val Ala Glu Leu Ala Ile Pro Thr Ala Glu 485 490495 Ser Gln Arg Ala Leu Val Gly Ser Gly Ile Glu Ala Gln Val Asn Gly 500505 510 Glu Arg Val Leu Ile Cys Ala Ala Gly Lys His Pro Ala Asp Ala Phe515 520 525 Thr Gly Leu Ile Asn Glu Leu Glu Ser Ala Gly Gln Thr Val ValLeu 530 535 540 Val Val Arg Asn Asp Asp Val Leu Gly Val Ile Ala Leu GlnAsp Thr 545 550 555 560 Leu Arg Ala Asp Ala Ala Thr Ala Ile Ser Glu LeuAsn Ala Leu Gly 565 570 575 Val Lys Gly Val Ile Leu Thr Gly Asp Asn ProArg Ala Ala Ala Ala 580 585 590 Ile Ala Gly Glu Leu Gly Leu Glu Phe LysAla Gly Leu Leu Pro Glu 595 600 605 Asp Lys Val Lys Ala Val Thr Glu LeuAsn Gln His Ala Pro Leu Ala 610 615 620 Met Val Gly Asp Gly Ile Asn AspAla Pro Ala Met Lys Ala Ala Ala 625 630 635 640 Ile Gly Ile Ala Met GlySer Gly Thr Asp Val Ala Leu Glu Thr Ala 645 650 655 Asp Ala Ala Leu ThrHis Asn His Leu Arg Gly Leu Val Gln Met Ile 660 665 670 Glu Leu Ala ArgAla Thr His Ala Asn Ile Arg Gln Asn Ile Thr Ile 675 680 685 Ala Leu GlyLeu Lys Gly Ile Phe Leu Val Thr Thr Leu Leu Gly Met 690 695 700 Thr GlyLeu Trp Leu Ala Val Leu Ala Asp Thr Gly Ala Thr Val Leu 705 710 715 720Val Thr Ala Asn Ala Leu Arg Leu Leu Arg Arg Arg 725 730 3 42 DNAArtificial Sequence Primer 3 ggatccaaag agtaaagaag aacaatgtcg actcctgacaat 42 4 25 DNA Artificial Sequence Primer 4 ggatccctct cctgcgcaac aatct25 5 18 DNA Artificial Sequence Primer 5 gagatgtcga gtctcgaa 18 6 23 DNAArtificial Sequence Primer 6 ctcgagcaca gtgtagtgac tgg 23

What is claimed is:
 1. A recombinant vector comprising a coding sequencefor a heavy metal-transporting P type ATPase, wherein the codingsequence is operably linked to and under the regulatory control of aplant-expressible transcription and translation regulatory sequence. 2.The recombinant vector according to claim 1, wherein the heavy metal isat least one selected from the group consisting of arsenic, antimony,lead, mercury, cadmium, chrome, tin, zinc, barium, nickel, bismuth,cobalt, manganese, iron, copper, vanadium.
 3. The recombinant vectoraccording to claim 1, wherein the P type ATPase is ZntA.
 4. Therecombinant vector according to claim 3, wherein the ZntA has an aminoacid sequence as given in SEQ ID NO:2.
 5. The recombinant vectoraccording to claim 1, wherein the coding sequence is ZntA-like heavyMetal pumping ATPase gene comprising a nucleic acid sequence sharing atleast about 50% homology with ZntA as given in SEQ ID NO:
 1. 6. Therecombinant vector according to claim 1, wherein the recombinant vectoris PBI121/zntA or pEZG.
 7. A transgenic plant, or parts thereof, eachtransformed with a recombinant vector of claim
 1. 8. The transgenicplant, or thereof according to claim 7, wherein the heavy metal is atleast one selected from the group consisting of arsenic, antimony, lead,mercury, cadmium, chrome, tin, zinc, barium, nickel, bismuth, cobalt,manganese, iron, copper, vanadium.
 9. A transgenic plant cell,transformed with a recombinant vector of claim
 1. 10. The transgenicplant cell according to claims 9, wherein the heavy metal is at leastone selected from the group consisting of arsenic, antimony, lead,mercury, cadmium, chrome, tin, zinc, barium, nickel, bismuth, cobalt,manganese, iron, copper, vanadium.
 11. A transgenic plant, stablytransformed with a recombinant vector of claim
 1. 12. The transgenicplant according to claim 11, wherein the heavy metal is at least oneselected from the group consisting of arsenic, antimony, lead, mercury,cadmium, chrome, tin, zinc, barium, nickel, bismuth, cobalt, manganese,iron, copper, vanadium.
 13. A transgenic plant, or parts thereof, eachtransformed with a recombinant vector of claim
 5. 14. The transgenicplant, or parts thereof according to claims 13, wherein the heavy metalis at least one selected from the group consisting of arsenic, antimony,lead, mercury, dadmium, chrome, tin, zinc, barium, nickel, bismuth,cobalt, manganese, iron, copper, vanadium.
 15. A transgenic plant cell,transformed with a recombinant vector of claim
 5. 16. The transgenicplant cell according to claims 15, wherein the heavy metal is at leastone selected from the group consisting of arsenic, antimony, lead,mercury, cadmium, chrome, tin, zinc, barium, nickel, bismuth, cobalt,manganese, iron, copper, vanadium.
 17. A transgenic plant, stablytransformed with a recombinant vector of claim
 5. 18. The transgenicplant according to claim 17, wherein the heavy metal is at least oneselected from the group consisting of arsenic, antimony, lead, mercury,cadmium, chrome, tin, zinc, barium, nickel, bismuth, cobalt, manganese,iron, copper, vanadium.
 19. A recombinant vector comprising a codingsequence for a heavy metal-transporting P type ATPase, ZntA of SEQ IDNO: 1; wherein the coding sequence is operably linked to and under theregulatory control of a plant-expressible transcription and translationregulatory sequence; and wherein the ZntA contains an approximately 100amino acid residue N-terminal extension domain, a first transmembranespanning domain, a second transmembrane spanning domain containing aputative cation channel motif CPX domain, a third transmembrane spanningdomain, a first cytoplasmic domain, a second cytoplasmic domain, and aC-terminal domain.
 20. A transgenic plant, or parts thereof, eachtransformed with a recombinant vector of claim
 19. 21. The transgenicplant, or parts thereof according to claims 20, wherein the heavy metalis at least one selected from the group consisting of arsenic, antimony,lead, mercury, cadmium, chrome, tin, zinc, barium, nickel, bismuth,cobalt, manganese, iron, copper, vanadium.
 22. A transgenic plant cell,transformed with a recombinant vector of claim
 19. 23. The transgenicplant cell according to claims 22, wherein the heavy metal is at leastone selected from the group consisting of arsenic, antimony, lead,mercury, cadmium, chrome, tin, zinc, barium, nickel, bismuth, cobalt,manganese, iron, copper, vanadium.
 24. A transgenic plant, stablytransformed with a recombinant vector of claim
 19. 25. The transgenicplant according to claim 24, wherein the heavy metal is at least oneselected from the group consisting of arsenic, antimony, lead, mercury,cadmium, chrome, tin, zinc, barium, nickel, bismuth, cobalt, manganese,iron, copper, vanadium.
 26. A recombinant vector comprising a codingsequence for a heavy metal-transporting P type ATPase, ZntA wherein thecoding sequence is operably linked to and under the regulatory controlof a plant-expressible transcription and translation regulatory; whereinthe ZntA contains an approximately 100 amino acid residue N-terminalextension domain, a first transmembrane spanning domain, a secondtransmembrane spanning domain containing a putative cation channel motifCPX domain, a third transmembrane spanning domain, a first cytoplasmicdomain, a second cytoplasmic domain, and a C-terminal domain; andwherein each of the domains of the coding sequence shares at least about50% homology with a same domain of SEQ ID NO:1.
 27. A transgenic plant,or parts thereof, each transformed with recombinant vector of claim 26.28. The transgenic plant, or parts thereof according to claims 27,wherein the heavy metal is at least one selected from the groupconsisting of arsenic, antimony, lead, mercury, cadmium, chrome, tin,zinc, barium, nickel; bismuth, cobalt, manganese, iron, copper,vanadium.
 29. A transgenic plant cell, transformed with a recombinantvector of claim
 26. 30. The transgenic plant, or parts thereof accordingto claims 29, wherein the heavy metal is at least one selected from thegroup consisting of arsenic, antimony, lead, mercury, cadmium, chrome,tin, zinc, barium, nickel, bismuth, cobalt, manganese, iron, copper,vanadium.
 31. A transgenic plant, stably transformed with a recombinantvector of claim
 30. 32. The transgenic plant according to claim 31,wherein the heavy metal is at least one selected from the groupconsisting of arsenic, antimony, lead, mercury, cadmium, chrome, tin,zinc, barium, nickel, bismuth, cobalt, manganese, iron, copper,vanadium.
 33. A method of producing a transgenic plant with enhancedresistance to heavy metals comprising: (a) preparing an expressionconstruct comprising a sequence encoding a heavy metal-transporting Ptype ATPase, operably linked to and under the regulatory control of aplant-expressible transcription and translation regulatory sequence; (b)preparing a recombinant vector harboring the expression construct; and(c) introducing the expression construct of the recombinant vector intoa plant cell or plant tissue to produce a transgenic plant cell ortransgenic plant tissue.
 34. The method of producing a transgenic plantaccording to claim 33, wherein the heavy metal is at least one selectedfrom the group consisting of arsenic, antimony, lead, mercury, cadmium,chrome, tin, zinc, barium, nickel, bismuth, cobalt, manganese, iron,copper, vanadium.
 35. The method of producing a transgenic plantaccording to claim 33, further comprising the step of: regenerating atransgenic plant from the transgenic plant cell or transgenic planttissue of step (c).